Team:Cambridge/Gibson/Protocol

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{{:Team:Cambridge/Templates/headerMinimalprototype}}
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{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Gibson Assembly: Protocols}}
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{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}
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The formal paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].
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The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].
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==Step 1) Design Primers==
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==Step 1: Design Primers==
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*If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.
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{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}
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<div style="float:right; clear:both">&nbsp;</div>
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*The standard way to do this is with PCR with specialised primers
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If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.
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*We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]
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The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]
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==Step 2: Order Primers==
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==Step 2) Order Primers==
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This step can take a while, so Gibson Assembly requires some planning ahead
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*This step can take a while, so Gibson Assembly requires some planning ahead
 
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==Step 3) PCR ==
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==Step 3: PCR ==
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PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer:
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==Step 4) Gibson Assembly==
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<div style="float:right; clear:both">&nbsp;</div>
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{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}
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<html>
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<style>
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table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}
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table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}
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</style>
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<div align="left">
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<table class="vistable" padding="0" cellspacing="0">
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*Prepare Master Mix
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<tr>
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=== Master mix ===
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<td>Step
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</td><td>Temp
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</td><td>Time
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</td></tr>
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<tr>
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<td>1:Initial Melting
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</td><td>98°C
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</td><td>30s
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</td></tr>
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<tr>
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<td>2:Melting
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</td><td>98°C
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</td><td>10s
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</td></tr>
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<tr>
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{|class="wikitable"
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<td>3:Annealing
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|-
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</td><td>T<sub>m</sub>°C
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|
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</td><td>15s
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|Volume/µl
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</td></tr>
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|-
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<tr>
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|Taq ligase (40u/µl)
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<td>4:Elongation
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|50
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</td><td>72
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|-
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</td><td>45s per kb DNA
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|5x isothermal buffer
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</td></tr>
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|100
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<tr>
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|-
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<td>5:GoTo step 2
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|T5 exonuclease (1u/µl)
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</td><td>
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|2
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</td><td>30 times
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|-
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</td></tr>
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|Phusion polymerase (2u/µl)
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<tr>
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|6.25
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|-
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|Nuclease-free water
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|216.75
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|-
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|
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|=375
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|}
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Master Mix is 1.33x concentrated
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<td>6:Final Elongation
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</td><td>72°C
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</td><td>7m30
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</td></tr>
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<tr>
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<td>7:Final Hold
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</td><td>4°C
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</td><td>∞
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</td></tr>
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*Add DNA to be ligated and master mix in volumetric ratio 1:3
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*Incubate for 1 hour at 50°C
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</table>
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</div>
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</html>
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The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.
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The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].
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==Step 4: Gibson Assembly==
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1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]
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2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3
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3) Incubate for 1 hour at 50°C
<i>e.g. If you were ligating two fragments (A and B) you could put:</i>
<i>e.g. If you were ligating two fragments (A and B) you could put:</i>
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{|class="wikitable"
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<html>
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|-
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<style>
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|<i>2.5µl</i>
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table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}
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|<i>fragment A</i>
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table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}
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|-
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</style>
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|<i>2.5µl</i>
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<div align="left">
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|<i>fragment B</i>
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<table class="vistable" padding="0" cellspacing="0">
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|-
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<tr>
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|<i>15µl</i>
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<td><i>2.5µl</i>
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|<i>Gibson Master Mix</i>
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</td><td><i>fragment A</i>
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|}
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</td></tr>
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<tr>
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<td><i>2.5µl</i>
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</td><td><i>fragment B</i>
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</td></tr>
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<tr>
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<td><i>15µl</i>
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</td><td><i>Gibson Master Mix</i>
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</td></tr>
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</table>
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</div>
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</html>
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==Step 5: Transformation==
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The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.
{{:Team:Cambridge/Templates/footer}}
{{:Team:Cambridge/Templates/footer}}

Latest revision as of 20:31, 26 October 2010

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